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Saraswati S
Abstract
Biology is in the midst of intellectual and experimental sea change. Essentially the
discipline is moving from being a largely data poor science to a data rich science.
transcriptomics and proteomics. Just as genomics is the omics for DNA sequence
analysis, metabolomics is the omics approach to understand cell and systems biology
Introduction
Metabolomics has been developed as one of the new ‘Omics’ joining genomics,
identify and quantify the complete set of metabolites present in a cell or tissue and
[2], [3].
to do so as quickly as possible and without bias It is a key aspect to phenotype;
[5]
functional genomics and may be the best and most direct measure of cellular
[6]
morphology
[1], [4].
physiological rate It defines metabolic phenotype thus is an important
[7].
biochemical manifestation, and useful tool for functional genomics Another
definition states that metabolome consists only of those native small molecules
reactions and that are required for maintenance, growth and normal function of a cell
[8].
“Omics” technologies are based on comprehensive biochemical and molecular
their respective classes of biomolecules- mRNAs, proteins and metabolites. While the
is the metabolome that represent the current status of the cell or tissue. To
feedback regulate gene and protein expression and mediate signal between
profiling technologies.
At the analytical level both the functional genomics and Metabolomics rely on
stagnated from 407 in 2005 to 406 in 2006. (Fig 1). The use of these “omics”
technologies in the biological research during the last 20 years is summarized in Fig.
Fig 1. Pubmed literature search results document the continuously growing research
[10]
transcriptomics
5
Metabolomics or Metabonomics
[1], [11] [12], [13], [14], [15], [16], [17], [18], [19], [20],
recent years and a volume on metabolic profiling
[21].
was published in 2003 Historically, metabolomics and metabonomics are
[22].
compared with GC/MS and NMR respectively L stands for plants and N stands for
animals. Before any further discussion a question, which arises, is what the
difference between metabonomics and metabolomics is, and when is the use of
either term appropriate? The possible answer might be whom you target as both the
terms may be appropriate in most cases and the distinctions are more a matter of
The concept of the metabolome has been in existence for years in the form of
[23], [24]
metabolic control theory and flux analysis and was routinely used in
[25],
publications which indicated the total metabolite pool; “metabolome” analysis
regulation. While not expressly defined, the term metabolomics was indicated by
[22]
Fiehn to be the "comprehensive and quantitative analysis of all metabolites. ..."
[26]
Nicholson coined Metabonomics in 1999 and defined as “the quantitative
naming convention problem is the fact that metabonomics and metabolomics have
elucidate the function of novel genes and play important role in future plant, nutrition
6
and health, drug toxicity etc. Metabolism is the key aspect of phenotype, hence
[28]
(i.e. by analyzing the metabolome functions may be assigned to respective gene .
Metabolites are the result of interaction of system’s genome with its environment and
are not merely end product of gene expression but also from part of regulatory
system in an integrated manner and thus can define biochemical and phenotype of a
[3]
cell or tissue . Thus its quantitative and qualitative measurement can provide a
broad view of biochemical status of organism; that can be used to monitor and
[1]
assess gene function .
Exhaustive work has been done on genomics, proteomics and transcriptomics, which
allowed establishing global and quantitating mRNA expression profile of cells and
[29]
tissues in species for which the sequence of all genes is known . Now question
which arises is why Metabolomics when transcriptome, genome and proteome are so
popular? Probable reason for this may be: any change in transcriptome and
[29]
biochemical phenotype and increase mRNA do not always correlated with
increased protein level. Translated protein may or may not be enzymatically active;
[2]
alteration in biochemical phenotype . Identification of mRNA and protein is indirect
and yield only limited information. Another reason might be: if quantification of
metabolite is known then long process like to know DNA protein sequence, micro
[30]
array, 2 D Gel Electrophoresis need not to be done . Thus, it is inferred that
[2]
metabolome provide the most functional information of Omics technology . Unlike
7
transcripts and proteome, metabolite shares no direct link with genetic code and is
and tissue. As such, metabolites do not readily tend themselves to universal methods
[31]
for analysis and characterization .
Metabolome data has twin advantage in systematic analysis of gene function; that
metabolites are functional cellular entities that vary with physiological content and
also the number of metabolites is far fewer than the number of genes or gene
may be achieved by comparing the change in cells metabolite profile that is produced
[32]
individually deleting genes of unknown function Strategies for identifying the
[33],
function of unknown genes on the basis of metabolomic data have been proposed
[34]
Silent phenotypes can be revealed by significant changes in concentration of
[33]
that does not participate directly in metabolism or its control . An advantage of
[2]
FANCY approach is that it assigns cellular rather than molecular function
[35], [36]
different genotypes or treated plants . Metabolic composition of a cell or tissue
influences the phenotype and it is the most appropriate choice for functional
genomics and to use the fluxes between metabolites as the basis for defining a
[37] [38]
metabolic phenotype is a matter for debate but there is increasing evidence,
for example from investigations of transgenic plants [39] that metabolomic analysis is a
defined, is greatly increased by the possibility of correlating the data with the
[40].
system-wide analysis of gene expression and protein content
[1]
exceed 200,000 different metabolites and therefore large-scale comprehensive
metabolite profiling meets its greater challenge. Metabolites are not linear polymers
diverse collection of molecule with widely varied chemical and physical properties.
[3].
profile all of metabolome simultaneously To find changes in metabolic network
that are functionally correlated with the physiological and developmental phenotype
[31].
of the cell, tissue or organism is the bottleneck of metabolomics If one general
extraction and analytical system is used it is likely that many metabolites will remain
[32].
in plant matrix and will not be profiled Analytical variance (the coefficient of
between plants of same species grown under identical or as near as possible identical
[2]
over which instrumental response as a function of analyte concentration is linear)
[14],
Metabolome analysis can be roughly grouped in to four categories which require
different methodologies for validation of results. For the study of primary effects of
metabolite target analysis and is mainly used for screening purpose. Sophisticated
methods for the extractions, sample preparation, sample clean ups, and internal
[22], [41].
references may be used, making it much more precise than other methods
origin and used for functional genomics, plant breeding and various diagnostic
was coined by Horning and Horning in 1970, defined as ‘quantitative and qualitative
analysis of complex mixtures of physiological origin’. It has been employed for the
[46]
. Only crude sample fractionation and clean-up steps are carried out [22], [41].
preparation and data acquisition aimed at including all class of compounds, with high
[14]
continued maturation of it, following objectives need to be achieved :
genomic strategies.
10
Metabolomics technologies:
[47]
Metabolites are chemical entities and be can be analyzed by standard tools of
chemical analysis much molecular spectroscopy and MS. For better resolution,
[47]
decides the use of different technologies and strategies . It is not yet technically
where GC first separates volatile and thermally stable compounds and then eluting
[48]
described as GOLD STANDARD : in spite of its biasedness against non-volatile,
high MW metabolites. Thermo-labile and large metabolites such as organic bis-, tri-
formation is required to eliminate undesirable slow and reversible slow and reversible
silylating reaction with carbonyl groups, whose products can be thermally labile. The
presence of water can result in breakdown of TMS esters, although extensive sample
drying and presence of exceeds silylating reagents can limit the process. Small
11
metabolites that are unresolved by GC. It can detect co-eluting peaks with peak,
apexes separated by less than 1s and also detect low-absorbance peaks co-eluting in
[35], [50]
Using gas chromatography-mass spectrometry (GC-MS) comprehensive
[2]
sugars, and 27 saponins in Medicago truncatula were identified . 326 distinct
[1]
compounds were identified in A. thaliana leaf extracts , further elucidating the
been investigated using fractionation techniques and about 100 compounds were
[51]
identified in rice grains via fractionation techniques by employing GC-MS . In GC-
[52]
determinations have been achieved by applying time-of-flight technology (TOF) .
[53]
measurement have however resulted in detection of over 1000 components from
plant leaf extracts at a throughput of over 1000 sample per month . Recent advance
[54]
is MSFACTs (Metabolomics Spectral Formatting and Conversion Tools) which
comprises of two tools, one for alignment of integrated chromatographic peak list
and another for extracting information from raw chromatic ASC II formatted data
files. Another recent advance is MET- IDEA (Metabolomics Ion-based Data Extraction
[55]
data files, which allows for more rapid biological insight .Over 300 metabolites
using GC-MS technology. Although, it has been shown that the number of detected
LC-MS techniques were developed employing soft ionization methods like electro
became both more sophisticated and more robust for daily use. More recently,
separation of the complex mixtures than it was attainable before. The objective of
this study was to develop LC-MS methods of analysis suitable for the plant
metabolomics studies, and to apply this for Arabidopsis and rice plants. LC-MS for
Capillary LC/MS using monolithic columns have been applied to metabolome profiling
[57]
of Arabidopsis . More than 1400 compounds from Arabidopsis leaf extract using a
[58]
quadrapole time-of-flight (QTOF) mass spectrometer were identified . The
[59]
problem, which arises with LC-MS, is ion suppression due to matrix effect ; that
[60]
can be circumvented by reducing the size of liquid droplets Due to this reason
[61]
capillary electrophoresis (CE) has been taken in to consideration . It is relatively a
new technology, which has been widely used for both targeted and non-targeted
[62]
analysis of metabolites . It has been used to analyze a variety of compounds
due to its high resolving power and small sample requirement with short analysis
13
[63], [64], [65], [66], [67], [68], [69]
time . CE is advantageous for measuring water-soluble
preparation is rapid and common to all type of compounds; every type of compound
[22], [41]
ionisable or ionic compounds Capillary electrophoresis-mass spectroscopy was
used to measure the intracellular levels of ionic and polar metabolites in bacterial
cells were developed [64], [ 73], [74]. Using CE-MS, 1692 metabolites were
[70]
identified in Bacillus subtilis extracts and CE-MS and CE-DAD, 88 main
[71
biosynthesis were measured in rice ].
[72], [73]
used in metabolomic analysis . NMR has low sensitivity than MS and suffers
recorded from cell suspensions, tissues, and even whole plants, as well as from
[74], [75]
extracts and purified metabolites . It offers an array of detection schemes that
can be tailored to the nature of the sample and the metabolic problem that is being
[75]
addressed . Thus analyzing the metabolite composition of a tissue extract,
a tissue are all possible by NMR. However, the nature of the NMR measurements that
are required for these tasks, particularly in relation to the hardware requirements,
[75]
the detection scheme, and the sensitivity of the analysis is very different . Third,
the natural abundance of some of the biologically relevant magnetic isotopes is low
with liquid chromatography can increase its efficiency by reducing the co-resonant
peaks and improving dynamic range. It has been reported that a combination of
HPLC-NMR spectroscopy with rudimentary data analysis has been employed for the
[78]
evaluation of metabolic changes in transgenic food crops . Using LC-NMR nearly
[78]
2700 analytes were detected in plant extracts . Directly coupled HPLC-NMR and
HPLC-NMR-MS has been used that allows rapid identification of metabolites with little
[79], [80]
sample preparation .
FTMS) has so far been used only in a handful of published studies into metabolomics
[85]
metabolites by chromatography .It has been used for characterization of lipo-
[86] [87]
oligosaccharides discovery of central nervous system agents and high
[88]
throughput screening of combinatorial libraries The extreme mass accuracy of the
technique, coupled to ultra high resolution of mass species means that thousands of
separations.
15
Application of Metabolomics
Plants are of pivotal importance to sustain life on Earth because they supply oxygen,
food, energy, medicines, industrial materials and many valuable metabolites. Plant
[ 89]
metabolites with this number set to rise to 200,000 . These plants metabolites are
each plant. Due to its possibility off making economical worthwhile discoveries,
plants have been the subject of many metabolomics research programs. It has been
[2]
expression and protein production . One of the first applications of the approach
was to genotype Arabidopsis thaliana leaf extracts. However, even after the
[91] [92]
completion of the genome sequencing of Arabidopsis and rice function of these
innovative way for identification of gene function for specific product accumulation in
[93], [94]
plants . Metabolomics can provide research a new tool to identify the functions
could lead to the engineering of the higher quality food or material producing plants.
plants in broader sense and possibly to unravel yet unknown changes in the plant
metabolome.
bear in quest to achieve fully personalizes and preventive health care. Bases of most
the rapid acquisition of large data sets. This makes it ideal as a screening tool,
particularly for human populations where there can be significant environmental and
[89]
dietary influences on tissue and biofluid ‘metabolomes’ . Metabolomics, in
conjunction with other "-omics" approaches, offers a new window onto the study of
[97] [98]
dystrophy , multiple sclerosis, schizophrenia , Amyotrophic lateral sclerosis
data exchange format. Large amount of data can be transmitted stored safely with
adequate curation and made available in convenient and supportive ways for
statistical analyses and datamining. To do this, well designed data standards are
what should be recorded for a transcriptome experiment. Possibly the most advanced
by pathways tool software developed by Peterskap’s group at SRI. The aim of AraCyc
expression data.
[103]
ArMET: ArMet is proposed framework for description of plant metabolomics
experiments and their result. It encompasses the entire experiment time line and
organizes it in into nines subunits termed components. In this data are specified by a
way of a core set of data items for each component. These core data provide a basis
for cross laboratory data exchange and datamining. This components based
approach for Armet provide a basis for definition of extension to core data to support
experiments and laboratories. ArMet compliant databases and data handling systems
support experiment with Arabidopsis thaliana and Solanum tuberosum. ArMet and
provides a formal data definition to support automatic data set comparison and
mining and development of system for data storage and exchange. It works in
synergy with laboratory information management system (LINS) and other existing
[104]
AraCyc is a database contains biochemical pathways of Arabidopsis, developed at
[105]
DOME is composed of various subsection: one counting details about
experimental design (metadata), another with raw data, another one with processed
data ( i.e. analysis result) and finally an ontology describing the known molecular
multiple statistical tool and visualize using a Brower for OME’s ( BROME).
[106]
MetaCyc is a database of non-reluctant, experimentally elucidated metabolic
pathways. MetaCyc comprised of near about 700 pathways from more than 600
recently began capturing qualitative data such as enzyme kinetics data. Goal of
can be converted to an XML file. From this XML file, it can be transferred to Gene
Gobi which uses the network in conjunction with statistical analysis of expression
data, to FC Modeler, which find cycles and pathways in the networks, visualizes and
models in combination with expression data and to MetNet, where networks can be
functionality to do statistical analyses (Gene Gobi) and versatile text mining (Path
binder A). This set of applications seeks how they interact in context of metabolic
networks. The MetNet software enables analyses of disparate data types (microarray,
network.
[108]
Map Man is a user driven tool that displays large datasets on to diagrams of
separate user-guided module. Editing existing module and creation of new categories
[109]
BioCyc is a collection of pathways/ genome database provide electron references
collection are recognized into tiers according to the amount of manual review.
20
[110]
BRENDA represents the most comprehensive information system on the enzymes
and metabolic information. The database contains data from atleast 83,000 different
literature references.
21
Future directions
Metabolomics is an emerging technology that has lot of scope and needs lots of
are need that can focus on the specific classes of small molecules so that remarkable
family of biomolecules of near limitless structural diversity unlike genes and proteins.
Increased sensitivity and high resolution tools combined with the exhaustive
in the number of instruments like NMR, MS, IR or any other technique will not solve
this problem, instead new technologies are needed and real jump in innovation or
even more important- better software technologies and curated and unified open
biochemical and signaling network is essential; for the systems biology and will help
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